Abstract
6-Pyruvoyltetrahydropterin synthase (PTPS) participates in tetrahydrobiopterin cofactor biosynthesis. We previously identified in a PTPS-deficient patient an inactive PTPS allele with an Arg(16) to Cys codon mutation. Arg(16) is located in the protein surface exposed phosphorylation motif Arg(16)-Arg-Ile-Ser, with Ser(19) as the putative phosphorylation site for serine-threonine protein kinases. Purification of recombinant PTPS-S19A from bacterial cells resulted in an active enzyme (k(cat)/K(m) = 6.4 x 10(3) M(-1) s(-1)), which was similar to wild-type PTPS (k(cat)/K(m) = 4.1 x 10(3) M(-1) s(-1)). In assays with purified enzymes, wild-type but not PTPS-S19A was a specific substrate for the cGMP-dependent protein kinase (cGK) type I and II. Upon expression in COS-1 cells, PTPS-S19A was stable but not phosphorylated and had a reduced activity of approximately 33% in comparison to wild-type PTPS. Extracts from several human cell lines, including brain, contained a kinase that bound to and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of cGMP stimulated phosphotransferase activity 2-fold. Extracts from transfected COS-1 cells overexpressing cGKII stimulated Ser(19) phosphorylation more than 100-fold, but only 4-fold from cGKI overexpressing cells. Moreover, fibroblast extracts from mice lacking cGKII exhibited significantly reduced phosphorylation of PTPS. These results suggest that Ser(19) of human PTPS may be a substrate for cGKII phosphorylation also in vivo, a modification that is essential for normal activity.
Highlights
Tetrahydrobiopterin (BH4)1 is an essential cofactor for the aromatic amino acid hydroxylases, i.e. phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase, as well as for all three nitric-oxide synthase isoforms [1]
Whereas the longterm regulation of BH4 biosynthesis involved an alteration in GTP cyclohydrolase I gene expression, the short-term increase was speculated to be due to phosphorylation of a biosynthetic enzyme capable of modifying BH4 metabolism
We addressed the question of which site in the human homohexameric pyruvoyltetrahydropterin synthase (PTPS) is phosphorylated, what effect such a post-translational modification exerts on protein activity in vivo, and which type of protein kinase (PK) might be responsible for the specific modification
Summary
Materials and Miscellaneous Methods—The mutagenic primer PTPS205 was synthesized on a Gene Assembler Plus DNA synthesizer (Amersham Pharmacia Biotech): 5Ј-GCGCTGAAGGCGATGCGGCGG3Ј; the underlined nucleotide indicates the mismatch that leads to the serine 19 to alanine exchange (S19A) in the corresponding human PTPS cDNA sequence [20]. Cells from confluent plates were washed twice with standard PBS buffer, and incubated for 5 min in ice-cold lysis buffer containing protease inhibitors (50 mM Hepes, pH 7.2, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 200 units/ml aprotinin). Steps beyond this point, unless indicated, were all performed at 4 °C. Samples were separated by 12.5% SDS-PAGE, stained with Coomassie Blue, dried, and autoradiographed
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